Background[unreadable] [unreadable] Neonatal-onset multisystem inflammatory disease (NOMID) is a rare autoinflammatory disorder characterized by urticarial rash, fever, aseptic meningitis, papilledema, sensorineural hearing loss, epiphyseal overgrowth of the long bones, growth retardation, and, in some cases, mental impairment. During an earlier reporting period we showed that NOMID is at the severe end of a spectrum of disorders caused by mutations in CIAS1, which encodes a protein, cryopyrin, that is a key regulator of interleukin 1 (IL-1) beta activation. The other two CIAS1-associated disorders are familial cold autoinflammatory syndrome (FCAS), which presents with cold-induced fevers and urticaria, and Muckle-Wells syndrome (MWS), which presents with fevers and urticaria not clearly linked to cold exposure, as well as sensorineural hearing loss, and, in some cases, amyloidosis. The cryopyrin mutations of FCAS, MWS, and NOMID are all associated with increased activation of IL-1-mediated inflammatory pathways. Aberrant IL-1 signaling also appears to play a role in the pathogenesis of familial Mediterranean fever (FMF), which is caused by mutations in a protein we discovered several years ago and named pyrin, and in adult-onset Stills disease, a disorder currently without identified genetic associations that presents with spiking fevers, evanescent salmon-colored rash, arthritis, and hepatosplenomegaly.[unreadable] [unreadable] In this project we utilize inhibitors of IL-1 signaling to study both the treatment and pathophysiology of these disorders. Two agents are currently under investigation. The first, anakinra, is an FDA-approved recombinant IL-1 receptor antagonist, binding to the IL-1 receptor without signaling. It is given as a daily subcutaneous injection. The second, IL-1 Trap, is an investigational longer-acting soluble IL-1 receptor fusion protein, binding IL-1 with high affinity and preventing it from triggering membrane receptors. It is given as a weekly subcutaneous injection. We have completed enrollment of 18 NOMID patients into the anakinra trial, and we are now evaluating long-term safety and efficacy. The followup period currently ranges from 2 to 3.5 years. The IL-1 Trap protocol is still open for enrollment, having accrued 8 of a target 15 patients with FCAS/MWS, adult-onset Stills disease, or colchicine-resistant FMF. In both studies clinical observations are complemented with careful laboratory analyses of gene expression, cytokine production, and other immunologic parameters. [unreadable] [unreadable] Results of the Last Year[unreadable] [unreadable] Trial of Anakinra in NOMID: We recently published data on the 6-month outcome of this trial. All 18 patients enrolled in the study had remarkable clinical and laboratory responses to anakinra. The median value of a disease-specific diary score dropped from 3.70 before treatment to 0.26 after six months of anakinra (p < .001). Similarly, the median erythrocyte sedimentation rate, C-reactive protein, and serum amyloid A decreased from 57.5 mm/hr, 5.29 mg/dl, and 174 mg/l before treatment to 16.0, 0.40, and 6, respectively, after 6 months of treatment (all p values < 0.001). In 12 patients for whom cerebrospinal fluid (CSF) could be evaluated, intracranial pressures dropped from a median of 297 before treatment to 197 at 3 months of treatment (p < 0.001). Magnetic resonance imaging (MRI) sequences of the inner ear showed a decrease or disappearance of cochlear enhancement in 13/17 patients at 3 months of treatment, while leptomeningeal enhancement improved in all 8 patients (of 15 imaged) in whom it was observed at baseline. During a drug withholding phase of the study, all 11 patients who participated developed rash, joint pain, headaches, fevers, and other clinical signs and symptoms that were present before their disease was treated with anakinra. In all patients laboratory markers of inflammation increased significantly during the flare period. Reinstitution of therapy produced the same immediate responses to anakinra as were seen with the first administration. Levels of interleukin 6 in serum and CSF decreased with treatment and again increased in the serum when the drug was withheld. Levels of anakinra in the CSF increased during therapy, suggesting drug penetration into the CSF. The patients cultured peripheral blood mononuclear cells spontaneously secreted high levels of IL-1 beta and had an exaggerated IL-1 beta response to lipopolysaccharide. Spontaneous and stimulated secretion of IL-1 beta decreased progressively with up to six months of therapy. Before treatment, transcript levels of several genes encoding proteins regulated by IL-1 beta were significantly increased, relative to controls. Anakinra decreased the expression of IL-1 beta and genes downstream of IL-1 beta, whereas expression increased during anakinra withdrawal. There were no drug-related serious adverse events. We are currently analyzing longer-term outcome data to determine whether we can maintain the clinical responses seen in the initial 6 months of the study. [unreadable] Laboratory studies in patients with NOMID indicate a hyperresponsiveness to cell stimulation with several TLR stimulants. Patient cells oversecrete IL-1 in response to LPS but also mount a response to much lower concentrations of stimulants than controls. This mechanism may shed light on the development of clnical flares in patients with NOMID. [unreadable] [unreadable] Trial of IL-1 Trap in Autoinflammatory Disease: We have currently recruited 5 patients with FCAS/MWS and 3 patients with adult Stills disease. All patients with FCAS/MWS have responded to therapy and we are in the process of evaluating these data. Preliminary results in adult-onset Stills disease indicate that two patients with Stills have a partial response and 2 patients had immediate and almost complete responses to IL-1 blockade. We are planning to perform microarray analysis of these patients to identify distinguishing pathophysiologic pathways in the different disease subsets. We also recruited one patient with FMF who had a partial response to Il-1 Trap and did not continue the 2nd year of treatment with Il-1 Trap. We have discontinued recruitment of patients with FMF.[unreadable] [unreadable] Conclusions and Significance[unreadable] [unreadable] The anakinra treatment protocol for NOMID demonstrates a crucial role for IL-1 in the pathophysiology of this illness, and indicates that, at least over a six-month period, this agent is safe and effective in the treatment of this devastating illness. Patients treated with this agent for longer periods of time have continued to show significant improvement in their symptoms, and we will observe over longer time intervals. In addition, a protocol to test whether early administration of an IL-1 blocking agent will prevent the development of major organ involvement in very young children has been implemented. [unreadable] Our trial of IL-1 Trap in patients with FCAS/MWS and adult-onset Stills disease suggests a role for this longer-acting IL-1 inhibitor in both diseases. Functional studies in patients with NOMID help us understand the mechnism of IL-1 oversecretion in patients with CIAS1 mutation positive and mutation negative NOMID and microarray studies are planned to dissect the IL-1-dependent pathways inhibited by anakinra and IL-1 Trap, possibly shedding light on additional aspects of the pathophysiology of NOMID and related disorders. In addition to the studies outlined above, over the next year we will conduct studies to better understatnd the role of CIAS1 mutations in keratinocyte cell lines from NOMID patients and on apoptosis in a chondrocyte cell line.